OXTRHigh stroma fibroblasts control the invasion pattern of oral squamous cell carcinoma via ERK5 signaling

The Pattern Of Invasion (POI) of tumor cells into adjacent normal tissues clinically predicts postoperative tumor metastasis/recurrence of early oral squamous cell carcinoma (OSCC), but the mechanisms underlying the development of these subtypes remain unclear. Focusing on the highest score of POIs (Worst POI, WPOI) present within each tumor, we observe a disease progression-driven shift of WPOI towards the high-risk type 4/5, associated with a mesenchymal phenotype in advanced OSCC. WPOI 4-5-derived cancer-associated fibroblasts (CAFsWPOI4-5), characterized by high oxytocin receptor expression (OXTRHigh), contribute to local-regional metastasis. OXTRHigh CAFs induce a desmoplastic stroma and CCL26 is required for the invasive phenotype of CCR3+ tumors. Mechanistically, OXTR activates nuclear ERK5 transcription signaling via Gαq and CDC37 to maintain high levels of OXTR and CCL26. ERK5 ablation reprograms the pro-invasive phenotype of OXTRHigh CAFs. Therefore, targeting ERK5 signaling in OXTRHigh CAFs is a potential therapeutic strategy for OSCC patients with WPOI 4-5.


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NCG triple immunodeficient mice, lacking T, B and NK cells (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt, from GemPharmatech Co. Ltd., Nanjing, China)were used (both genders) for the orthotopic xenograft at 4-6 weeks of age and housed in ultraclean barrier facilities. Both gender of Fsp1-Cre mice and the Oxtr-flox/flox mouse strain was established in the Genetically Engineered Mouse Facility at GemPharmatech Co. Ltd. n(Nanjing, China). The Oxtrfl/fl S100a4cre mouse strain was generated by crossing the Fsp1-Cre (B6/JGpt-Tg(S100a4-CreERT2-PolyA)3/Gpt) and Oxtrflox/flox mouse strains (B6/JGpt-Oxtrem1Cflox/Gpt) with genotypic and phenotypic characterization. The housing conditions for the mice were as follows: 12h light/12 h darkness; temperature was 72 degrees Fahrenheit; and humidity was 40-50%. Mice were sacrificed by cervical dislocation at the indicated time points or when the largest tumor exceeded 1500 mm3 in subcutaneous models or 40 mm3 in orthotopic model (no mice exceeded these limits). In some cases, this limit has been reached the

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All male and female patients (26-88 year old, Asian) in this study diagnosed with primary OSCC were confirmed by hematoxylin and eosin staining of tumour biopsy.
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To compare CAF phenotypes in different groups, cells were resuspended in PBS containing 1% FBS and stained with fluorescent-conjugated antibodies to detect CD29, FAP or PDGFR-". For cell sorting in OSCC tissues, single cell suspensions were prepared from OSCC tissues via brief trypsinization. This was followed by forward and side scatter flow cytometry gating to exclude cell debris and dead cells by DAPI positive staining. Cells were stained with fluorescent-conjugated antibodies to detect EpCAM, CD45, CD31, PDGFR-" or FAP positive cell subpopulations. To facilitate isolation of OXTR high or low- Correction expressing CAFs, live single cells were labelled with APC/PE-conjugated anti-OXTR and sorted according to the OXTR fluorescence intensity, which was verified at the protein level by Western blot.